The present invention relates generally to methods for using breath carbon monoxide (xe2x80x9cCOxe2x80x9d), more particularly end-tidal carbon monoxide (xe2x80x9cETCOcxe2x80x9d) concentration measurements, to predict the occurrence of various pathological conditions during pregnancy as well as methods for using such measurements to determine the actual onset of such conditions.
A. Pregnancy Induced Hypertension, Preeclampsia and the HELLP Syndrome
Pregnancy-induced hypertension (xe2x80x9cPIHxe2x80x9d) is a common medical complication of pregnancy which encompasses a group of disorders including preeclampsia (xe2x80x9cPETxe2x80x9d). PET is primarily a disease of the last trimester of pregnancy and is the most common medical complication of pregnancy. It has a reported incidence ranging between 5% and 10% depending on the population demographics.1 As the pathogenesis of these disorders is unclear, prediction and prevention remain an elusive goal.
PET is more common in a woman""s first pregnancy and in women whose mothers or sisters had PET. The risk is also higher in women carrying multiple babies, in teenagers, and in women older than 40. Other women at risk include those with high blood pressure or kidney disease before becoming pregnant. The exact cause of PET is unknown. Symptoms of PET often include high blood pressure and proteinuria, and, in severe cases, headaches, blurring of vision and seizures. PET is often referred to by care providers as the xe2x80x9cgreat masqueraderxe2x80x9d given its ability to mimic many other conditions such as the flu, a kidney infection or gallbladder disease.
End-organ damage, especially to the kidneys and liver, may result if the condition is not recognized early enough, and the condition can be life-threatening. Consequences for the late-term fetus include diminished placental blood flow, and subsequent wasting and asymmetrical growth of the fetus. At present, the best cure for the disease is delivery of the preterm baby followed by intensive neonatal care for the baby and intensive surgical care for the mother.
No single test is currently known which can diagnose PET. A pregnant woman""s blood pressure is generally checked at each doctor""s visit and a surge in blood pressure can be an early sign of PET. A urine test can determine if protein has been excreted in the urine. Certain blood tests may also indicate PET. When signs of PET appear, a doctor should watch the pregnant woman closely, possibly even daily, for rises in blood pressure, swelling, or urine excretion.
PET, and especially its severe variant, the HELLP (Hemolysis, Elevated Liver Enzymes and Low Platelets) Syndrome, a syndrome with a reported incidence in PET of between 2% and 12%, are frequently misdiagnosed at initial presentation.2 The pathogenesis of the HELLP Syndrome remains unclear. Hemolysis, defined as the presence of microangiopathic hemolytic anemia, is the hallmark of the HELLP Syndrome.3 The diagnosis of hemolysis in pregnant women is currently based on an abnormal peripheral blood smear, increased bilirubin  greater than 1.2 mg/dl and an increased lactic dehydrogenase  greater than 600 IU/L.4 However, it is difficult to identify women at risk for HELLP Syndrome as severe hypertension is not a constant or even a frequent finding in the HELLP Syndrome.5 Early diagnosis is critical because the morbidity and mortality rates associated with the severe forms of this disease have been reported to be as high as 25 percent.6 Neonatal morbidity and mortality are also high in these pregnancies.7 
Since a delay in diagnosing PET can be fatal8, a better means of identifying women destined to develop PET is desirable. An accurate predictive test may allow timely transfer of patients to centers where adequate intervention and treatment could be promptly provided. Thus far, no test exists that satisfies the criteria for a suitable screening.9 
B. Premature Labor
Premature labor remains an important problem facing modem care-givers of pregnant women, demanding tremendous costs for the care of prematurely born infants. Preterm birth is the major cause of perinatal morbidity and mortality in the world. Prematurity is responsible for 75% of infant deaths and 50% of the long-term neurological handicaps, including cerebral palsy, blindness, deafness, and slow development. The survival rate of neonates is improved by 2% per day from the 23rd to the 26th week of pregnancy (i.e., from 16% at 23 weeks to 57% at 26 weeks), reaching an 80% survival rate at 28 weeks, and greater than 90% by 30 weeks of gestation. Therefore, any treatment that prevents or delays premature birth will profoundly reduce neonatal mortality and morbidity rates. However, the use of premature delivery depends on accurate early identification of premature uterine contractions (xe2x80x9cPMCxe2x80x9d).10 
Recent attention has focused on the part that NO might play in maintaining myometrial (uterine smooth muscle) quiescence during pregnancy. CO, like NO, stimulates soluble guanylyl cyclase, thereby raising intracellular levels of cGMP in smooth muscle to produce relaxation. It has been suggested that the L-arginine-NO system may contribute to uterine quiescence during gestation and the initiation of labor at term.11 
CO may similarly suppress myometrial contractility. The expression of large increases during pregnancy of heme oxygenase (xe2x80x9cHOxe2x80x9d), which catalyzes the degradation of heme to biliverdin and CO, has recently been demonstrated in the human myometrium.12 Furthermore, Acevedo and Ahmed have recently shown that induction of HO produces CO that limits uterine contractility in pregnant myometrium indicating a role for the HOxe2x80x94CO-cGMP pathway in the maintenance of the quiescent state of the uterus during pregnancy.13 A subsequent study did not support an up-regulation of HO during pregnancy and the data was inconsistent with a major role for CO in human myometrial quiescence.14 
It has recently been found in an analysis of heme oxygenase-1 (xe2x80x9cHO-1xe2x80x9d) immunoreactive protein of rat uterus and placenta that, during pregnancy, the expression of HO increases, up to day 16, then decreases toward delivery.15 Similarly, in rat tissues during pregnancy it has been found that in the myometrium and placenta, HO-1 mRNA levels increase during pregnancy, up to day 16, then decrease towards delivery.
C. Intrauterine Growth Retardation
Intrauterine growth retardation (xe2x80x9cIUGRxe2x80x9d) is an important cause of perinatal morbidity and mortality. The pathophysiology that precedes the development of IUGR remains incompletely understood. The importance of the placental blood flow to the growing fetus is obvious. The possible role of HO and its by-product CO in the regulation of blood pressure and blood flow have only been realized over the last few years.
Recently, a case of heme oxygenase-1 (xe2x80x9cHO-1xe2x80x9d) deficiency was presented.16 The patient had a complete loss of exon-2 of the maternal allele and a two-nucleotide deletion within exon-3 of the paternal allele. This child had severe growth retardation, hemolytic anemia, low bilirubin levels, elevated thromomodulin and Von Wilebrand factor as well as iron deposition in the liver and kidney. This presentation was very similar to that observed in the HO-1 null mutant mice. In normal gestation the HO-1 enzyme is seen at high levels in the neonatal and fetal rat lung and liver, compared to adults.17 
A current study found that HO expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.18 Furthermore, the results suggested a role for CO in placental function, trophoblast invasion, and spiral artery transformation.19 
D. The Effect of carbon monoxide in pregnancy
Cigarette smoking during pregnancy has been recognized for over three decades to be associated with reduced risks of PET.20 This paradoxical effect of exposure to tobacco during pregnancy, where smoking reduces the incidence of PET, but increases the perinatal morbidity and mortality and results in a well recognized health hazard to both the mother and her newborn, has long puzzled investigators.21 A recent systematic review of the existing evidence found that the risk of PET in pregnant women who smoked was 32% lower than that among nonsmoking pregnant women.22 Furthermore, pooled data from cohort and case-control studies showed that this inverse association was dose-related and remarkably consistent across studies conducted in various populations and countries.23 A current study that measured urinary cotinine in order to assess tobacco exposure confirmed the reduced risk of developing PET with cigarette smoking.24 Using data from the Collaborative Perinatal Project, it was recently demonstrated that smoking is associated not only with a reduced risk of hypertension during pregnancy, but also with a protective effect that appeared to continue even after cessation of smoking.25 
Despite the accumulating data suggesting an inverse association between cigarette smoking and the risk of PET, little light has so far been shed on the pathophysiology behind the apparent protective role of smoking. Smokers are exposed to high concentrations of CO which is a simple diatomic gas molecule that shares some of the physiochemical properties of nitric oxide (xe2x80x9cNOxe2x80x9d). CO, like NO, stimulates soluble guanylyl cyclase and thereby raises intracellular levels of cyclic guanosine monophosphate (xe2x80x9ccGMPxe2x80x9d) in vascular smooth muscle to produce endothelium-dependent arterial relaxation.26 It is now recognized that CO may play a key role in the regulation of placental hemodynamics.27 Platelet aggregation may also be prevented by CO generation.
It has recently been suggested that PET may reflect a state of impaired NO synthesis due to failure of the vasodilatation and decreased vascular reactivity. The similarity in the mechanisms of action of NO and CO in endothelium-dependent arterial relaxation suggest that CO formation could also have a contributory role in the pathogenesis of PET.28 This is supported by recent observations that HO, which catalyzes the degradation of heme to biliverdin and CO, is expressed more intensely in the placenta, umbilical cord, and myometrium of preeclamptic patients compared with normal pregnant patients.29 
Although possible roles of CO in pregnancy have recently been recognized, all previous studies have been based on laboratory analysis of tissue samples. Thus, these methods are difficult to implement and are time consuming and discomforting to the pregnant patients. Prior to applicant""s discovery disclosed herein, it is believed that nobody has ever attempted to look for clinical applications to determine the CO concentration measuring a pregnant patient""s breath. Nor was it known or even recognized to be useful to examine whether breath CO or ETCOc measurements could be used as a diagnostic and predictive tool in pregnant women.
E. End-Tidal Concentration of Carbon Monoxide and Measurement Thereof
The concentration of CO in the end-tidal breath, i.e., the gas that is last expelled in each breath, is presumed to be at equilibrium with the concentration in the blood. This is because the end-tidal breath contains predominantly, if not exclusively, the gas expelled from the alveoli in the lungs, which gas was within the alveoli for a time generally sufficient to equilibrate with the blood.
End-tidal carbon monoxide (xe2x80x9cETCOxe2x80x9d) concentrations, sometimes referred to as alveolar concentrations, can be measured in a variety of ways. A preferred methodology is described in U.S. Pat. No. 5,293,875, the disclosure of which is incorporated herein by reference, in which a breath sample is continuously drawn from the patient""s breath stream and directed to a fast-responding carbon dioxide (CO2) sensor and a slower responding CO sensor. The signals from the CO2 sensor, the CO sensor and a measurement of inhaled CO concentration are used to calculate the end-tidal CO concentration.
There are numerous other methods by which an end-tidal breath sample can be collected and analyzed for CO including the basic method of having the patient breathe through a tube connected to a three-way valve. During the first portion of the breath, which is non-alveolar, the valve is set so that the exhaled breath is directed to a chamber or discharged to the ambient environment. Towards the end of the exhalation, the valve can be manually switched so that the breath is directed into a sampling container or bag. The breath collected in the container can then be analyzed for the end-tidal CO concentration.
This basic method can be improved by automating the actuation of the three-way valve based upon a variety of signals including CO2 concentration, chest-wall movement, exhaled breath volume (described in U.S. Pat. No. 3,622,278), exhaled breath temperature, or changes in exhaled breath temperature (described in U.S. Pat. No. 4,248,245).
An alternative-method to obtain an end-tidal sample is to place the end of a syringe into a patient""s breath stream and manually pull the plunger of the syringe back during the final portion of the exhalation phase so that an end-tidal sample is drawn into the syringe. This method can be improved by automating the movement of the syringe plunger using a variety of control signals including exhaled CO2 concentration, chest-wall movement, exhaled breath volume or exhaled breath temperature (described in U.S. Pat. No. 4,220,162).
Still another way to obtain an end-tidal breath sample is to have the patient exhale through a reservoir which is much smaller than the tidal volume of the breath and has both inlet and outlet ports. The first portion of the breath is discharged to the ambient environment, while the end-tidal portion is trapped in the reservoir by either one-way check valves (described in U.S. Pat. No. 3,858,573), or by manually sealing the ports of the reservoir (described in U.S. Pat. No. 5,211,181).
The present invention relates to a method for using breath CO or ETCOc measurements to detect various pathological conditions in pregnant women including PIH, PET, PMC, and IUGR. The invention has further applications in the estimation of the severity of these abnormal conditions of pregnancy and in monitoring the response of such conditions to treatment.
The present invention provides a method for early detection of pathological conditions in pregnancy by measuring breath CO or ETCOc and can be applied in a hospital, clinic, or physician""s office. The method can utilize a device such the Natus(copyright) CO-Stat(copyright) End Tidal Breath Analyzer (Natus Medical Inc., San Carlos, Calif.) to perform non-invasive, simple and rapid, automatic sampling and analysis of end expiratory air without the requirement for laboratory analysis or highly trained personnel. Thus, the device and method are capable of providing on-the-spot instant results vital for rapid treatment of such serious conditions of pregnancy.
Two separate studies were conducted. In the first study, ETCOc measurements were prospectively performed on three study groups of women in three separate medical centers (the xe2x80x9cPIH/PET studyxe2x80x9d). The study groups included 52 women with PIH or symptoms of PET classified according to the American College of Obstetrics and Gynecology criteria (the xe2x80x9cPIH/PET test groupxe2x80x9d).30 The control groups included 42 first and 63 third trimester normotensive pregnant women and 46 non-pregnant women.
In the second study, ETCOc measurements were made in 55 nonsmoking, healthy pregnant women (the xe2x80x9cPMCxe2x80x9d studyxe2x80x9d). The study group included 10 women with PMC during the second half of their pregnancy (the xe2x80x9cPMC groupxe2x80x9d), 13 women in active labor at term and 32 pregnant mothers at matched gestational ages not experiencing uterine contractions.
It is an object of the present invention to utilize breath CO or ETCOc measurements to identify, either directly or indirectly, fundamental disturbances in normal regulation of placental and myometrial function, thereby allowing both prediction and early detection of various severe disorders of normal physiology associated with the major morbidities of pregnancy. Particularly, the method would allow detection of pregnant patients at risk for PIH, PET, the HELLP Syndrome, PMC, and IUGR.
Another object of the present invention is to allow early detection of these various pregnancy related conditions utilizing a safe, inexpensive, non-invasive diagnostic tool which can be operated with little training in a plethora of locations which are easily accessible to regnant women. Moreover, obtaining breath CO or ETCOc measurements advantageously avoids the hazards associated with the handling of blood samples.
Further objects and advantages afforded by the present invention will be apparent from the detailed description hereinbelow.